1. Field of the Invention
This invention concerns methods for engineering anti-tissue factor (anti-TF) antibodies, especially those having enhanced anticoagulant potency. The invention further concerns anti-TF antibodies, methods and means for producing them, compositions comprising the antibodies and their use in the diagnosis, management, prevention and treatment of various diseases and disorders.
2. Description of the Related Art
A. Tissue Factor
Tissue factor (TF) is the receptor for coagulation factor VIIa (FVIIa) and the zymogen precursor factor VII (FVII). Native human TF (hTF) is a 263 amino acid residue glycoprotein composed of an extracellular domain (residues 1 to 219), a single transmembrane domain (residues 220-242), and a short cytoplasmic domain (residues 243 to 263) (Fisher et al., [1987] Thromb. Res. 48:89-99, Morrissey et al., [1987] Cell 50:129-135). The TF extracellular domain is composed of two immunoglobulin-like fibronectin type III domains of about 105 amino acids each (Huang et al., [1998] J. Mol. Biol. 275:873-894). Each domain is formed by two anti-parallel xcex2-sheets with Ig superfamily type C2 homology.
The protein interaction of FVIIa with TF is mediated entirely by the TF extracellular domain (Muller et al., [1994] Biochem. 33:10864-10870; Gibbs et al., [1994] Biochem. 33:14003-14010; Ruf et al. [1994] Biochem. 33:1565-1572) which has been expressed in E. coli, cultured Chinese Hamster Ovary (CHO) cells and Saccharomyces cerevisiae (Waxman et al., [1992] Biochemistry 31:3998-4003; Ruf et al., [1991] J. Bio. Chem. 25 266:2158-2166 and Shigematsu et al., [1992] J. Biol. Chem. 267:21329-21337). The crystal structures of hTF extracellular domain and its complex with active site inhibited FVIIa have recently been determined by x-ray crystallography (Harlos et al., [1994] Nature 370:662-666; Muller et al., [1994] Biochemistry 33:10864; Muller et al., [1996] J. Mol. Biol. 256:144-159; Banner et al., [1996] Nature 380:41-46).
The hTF extracellular domain has also been extensively characterized by alanine scanning mutagenesis (Kelley et al., [1995] Biochemistry, 34:10383-10392; Gibbs et al., [1994] supra; Ruf et al., [1994] supra). Residues in the area of amino acids 16-26 and 129-147 contribute to the binding of FVIIa as well as the coagulant function of the molecule. Residues Lys20, Trp45, Asp58, Tyr94, and Phe140 make a large contribution (1 kcal/mol) to the free energy (xcex94G) of binding to FVIIa (Kelley et al., (1995) supra). Substitution of Lys20 and Asp58 with alanine residues leads to 78- and 30-fold reductions in FVIIa affinity respectively (Kelley et al., [1995] supra). A set of 17 single-site mutants at other nearby sites that are in contact with FVIIa result in modest decreases in affinity (xcex94xcex94G=0.3-1.0 kcal molxe2x88x921). Mutations of TF residues Thr17, Arg131, Leu133 and Val207, each of which contact FVIIa in the crystal structure, have no effect on affinity for FVIIa. Lys15Ala and Tyr185Ala mutations result in small increases in affinity (xcex94xcex94G=xe2x88x920.4 kcal molxe2x88x921) (Kelley et al., [1995] supra). The 78-fold decrease in affinity imposed by the alanine substitution of Lys20 in hTF can be reversed by substituting a tryptophan for Asp58 (Lee and Kelley, [1998] J. Biol. Chem. 273:4149-4154).
Residues in the area of amino acids 157-168 contribute to the procoagulant function of TF-FVIIa (Kelley et al., [1995] supra; Ruf et al., [1992] J. Biol. Chem. 267:22206-22210) but are not important for FVII/FVIIa binding. It has been shown that lysine residues 165 and 166 are important to TF cofactor function but do not participate in FVIIa complex formation (Roy et al., [1991] J. Biol. Chem. 266:22063; Ruf et al., [1992] J. Biol. Chem. 267:6375). Lysine residues 165 and 166 are located on the C-terminal fibronectin type III domain of TF on the opposite surface of the molecule from residues found to be important for FVIIa binding on the basis of mutagenesis results (Kelley et al., (1995) supra). Alanine substitution of these lysine residues results in a decreased rate of FX activation catalyzed by the TF-FVIIa complex (Ruf et al., (1992) supra). The Lys165Ala-Lys166Ala variant (hTFAA) comprising residues 1-219 of hTF (sTF) inhibits the extrinsic pathway of blood coagulation in vitro through competition with membrane TF for binding to FVIIa. In a rabbit model of arterial thrombosis the variant partially blocks thrombus formation without increasing bleeding tendency (Kelley et al., (1997) Blood 89, 3219-3227). However, high doses of the variant are required for the antithrombotic effect, in part because FVIIa binds to cell surface TF approximately 1000-fold more tightly than to sTF (Kelley et al. (1997) supra). The greater apparent affinity is due to interaction of the FVIIa xcex3-carboxyglutamic acid-containing (Gla) domain with phospholipid.
TF is expressed constitutively on cells separated from plasma by the vascular endothelium (Carson, S. D. and J. P. Brozna, [1993] Blood Coag. Fibrinol. 4:281-292). Its expression on endothelial cells and monocytes is induced by exposure to inflammatory cytokines or bacterial lipopolysaccharide (Drake et al., [1989] J. Cell Biol. 109:389). Upon tissue injury, the exposed extracellular domain of TF forms a high affinity, calcium dependent complex with FVII. Once bound to TF, FVII can be activated by peptide bond cleavage to yield serine protease FVIIa. The enzyme that catalyzes this step in vivo has not been elucidated, but in vitro FXa, thrombin, TF-FVIIa and FIXa can catalyze this cleavage (Davie, et al., [1991] Biochemistry 30:10363-10370). FVIIa has only weak activity upon its physiological substrates FX and FIX whereas the TF-FVIIa complex rapidly activates FX and FIX.
The TF-FVIIa complex constitutes the primary initiator of the extrinsic pathway of blood coagulation (Carson, S. D. and Brozna, J. P., (1993) Blood Coag. Fibrinol. 4:281-292; Davie, E. W. et al., [1991] Biochemistry 30:10363-10370; Rapaport, S. I. and L. V. M. Rao, [1992] Arterioscler. Thromb. 12:1111-1121). The complex initiates the extrinsic pathway by activation of FX to Factor Xa (FXa), FIX to Factor IXa (FIXa), and additional FVII to FVIIa. The action of TF-FVIIa leads ultimately to the conversion of prothrombin to thrombin, which carries out many biological functions (Badimon, L. et al., [1991] Trends Cardiovasc. Med. 1:261-267). Among the most important functions of thrombin is the conversion of fibrinogen to fibrin, which polymerizes to form a clot.
The involvement of this plasma protease system has been suggested to play a significant role in a variety of clinical manifestations including arterial and venous thrombosis, septic shock, adult respiratory distress syndrome (ARDS), disseminated intravascular coagulation (DIC) and various other disease states (Haskel, E. J. et al., [1991] Circulation 84:821-827; Holst, J. et al., [1993] Haemostasis 23 (suppl. 1):112-117; Creasey, A. A. et al., [1993] J. Clin. Invest. 91:2850-2860; see also, Colman R. W. [1989] N. Engl. J. Med 320:1207-1209; Bone, R. C. [1992] Arch. Intern. Med. 152:1381-1389). Overexpression and/or aberrant utilization of TF has been linked to the pathophysiology of both thrombosis and sepsis (Taylor et al., [1991] Circ. Shock 33:127; Warr et al., [1990] Blood 75:1481; Pawashe et al., [1994] Circ. Res. 74:56). TF is expressed on cells found in the atherosclerotic plaque (Wilcox et al., [1989] Proc. Natl. Acad. Sci. U.S.A. 86:2839). Additionally, TF has been implicated in tumor metastasis (Bromberg et al., [1995] Proc. Natl. Acad. Sci. USA, 92:8205).
B. Anti-Tissue Factor Antibodies
Monoclonal antibodies in humanized or chimaeric forms are successfully used to treat a variety of diseases (Vaswani and Hamilton, [1998] Ann. Allergy Asthma Immunol. 81: 105-119; Vaughan et al., [1998] Nature Biotechnology 16: 535-539).
Antibodies reactive with hTF have been described (Tanaka et al., [1985] Throm. Res. 40:745-756; Tanaka et al., [1986] Chem. Abstracts, 104:366:4921 1z; Morrissey et al., [1988] Throm. Res. 52:247-260; U.S. Pat. No. 5,223,427; Ruf et al., [1992] J. Crystal Growth 122:253-264; Huang et al., [1998] 275:873-894). Anti-TF monoclonal antibodies have been shown to inhibit tissue factor activity in various primate and non-primate species (Morrissey et al., [1988] supra; Huang et al. [1998] supra). Neutralizing anti-TF monoclona antibodies have been shown to prevent death in a baboon model of sepsis (Taylor et al., [1991] Circ. Shock 33:127), and attenuate endotoxin-induced DIC in rabbits (Warr et al., [1990], Blood 75:1481).
Inhibition of TF initiated blood coagulation by antibodies reactive with tissue factor has been proposed as a therapeutic modality (European Patent No. 0 266 993 B1), and the use of antibodies that specifically recognize TF at the site of thrombogenesis is currently viewed as a promising strategy for treating various thrombotic disorders. In fact, in vivo studies with anti-TF monoclonal antibodies demonstrated efficient anticoagulant activities (Levi et al., [1994] J. Clin. Invest. 93, 114-120; Taylor et al., [1991] Circulatory Shock 33, 127-134; Himber et al., [1997] Thromb Haemostasis 78, 1142-1149; Pawashe et al., [1994] Circ. Res. 74, 56-63; Ragni et al., [1996] Circulation 93, 1913-1918; Jang et al., [1992] Arterioscl. Thromb. 12, 948-954; Thomas et al., [1993] Stroke 24, 847-854; Golino et al., [1996] Nature Med. 2, 35-40). The use of a CDR-grafted anti-hTF antibody has been described for the attentuation or prevention of tissue factor mediated coagulation (International Publication No. WO 96/40921).
However, the precise TF binding sites of the antibodies used in the foregoing in vivo studies, with the exception of the antibody used by Levi et al., supra, are not known. The location of the antibody binding epitope may represent a critical factor in determining the inhibitory potencies of antibodies, because the cofactor function of TF involves several defined regions of the TF molecule. As a cofactor for factor VIIa (FVIIa), the cell surface exposed TF immobilizes FVII/FVIIa to the cell membrane thereby stabilizing the overall conformation of FVIIa (Waxman et al., [1993] Biochemistry 32, 3005-3012). The binding to TF also leads to the correct spatial orientation of the catalytic domain and the positioning of the active site in respect to the phospholipid membrane (McCallum et al., [1997] J. Biol. Chem. 272, 30160-30166; Banner et al., [1996] Nature 380, 41-46). Most of the TF-FVIIa contact surface area is provided by the FVIIa light chain interaction with TF. A smaller, yet critical contact surface lies between the N-terminal TF domain and the FVIIa catalytic domain. This contact is thought to play a main role in the enhancement of catalysis towards small synthetic as well as to macromolecular substrates (Dickinson et al., [1996] Proc. Natl. Acad. Sci. USA 93, 14379-14384; Dickinson and Ruf, [1997] J. Biol. Chem. 272, 19875-19879). In addition, TF participates in direct interaction with substrates (Huang et al., [1996] J. Biol. Chem. 271, 21752-21757) via residues K165 and K166 (Huang et al., supra; Ruf et al., [1992] J. Biol. Chem. 267, 6375-6381; Roy et al., [1991] J. Biol. Chem. 266, 22063-22066; Kelley et al., [1995] Biochemistry 34, 10383-10392), and neighboring residues (Ruf et al., [1992] J. Biol. Chem. 267: 22206-22210) in the C-terminal domain of TF. To add to this complex cofactor-enzyme-substrate interplay, recent observations suggested that the xcex3-carboxyglutamic acid-rich (Gla) domain of FVIIa contributes to substrate interaction (Huang et al., [1996] supra; Ruf et al., [1991] J. Biol. Chem. 266, 15719-15725; Martin et al., [1993] Biochemistry 32, 13949-13955; Ruf et al., [1999] Biochemistry 38, 1957-1966). Thus, anti-TF antibodies by virtue of their epitope location may interfere with one or several of these TF-mediated processes, which could translate into differences in their anticoagulant effectiveness. Such antibody epitope-dependent differences in potencies could be exacerbated under non-equilibrium conditions, which most likely prevail under therapeutic conditions. In this setting, antibody and the substrates circulating in blood would simultaneously interact with exposed TF.
In view of the limited characterization of most anti-TF antibodies known in the art, and the complexity of the mechanism by which TF exerts its thrombotic activity, it has so far been impossible to reliably engineer anti-TF antibodies with enhanced anticoagulant potency.
It is an objective of the present invention to determine which characteristics of anti-TF antibodies have the most profound effect on their anticoagulant properties. It is another objective, to design anti-TF antibodies with enhanced anticoagulant potency.
The present invention is based in part on the experimental finding that potency differences between various anti-TF antibodies can be explained by the location of the TF epitopes to which the antibodies bind and consequently, by the particular mode of inhibition. Anti-TF antibodies which bind to an epitope overlapping with the C-terminal macromolecular substrate-binding region of TF, and thus interfere with the TF-substrate interaction, are the most potent anticoagulant agents. This finding permits, for the first time, the purposeful design of anti-TF antibodies with high potency to treat or inhibit thrombosis.
Accordingly, one aspect of invention concerns a method for identifying anti-tissue factor (anti-TF) antibodies with enhanced anticoagulant potency, comprising (a) subjecting a plurality of anti-TF antibodies to epitope mapping, and (b) selecting antibodies binding to an epitope comprising at least part of the C-terminal macromolecular substrate-binding region of tissue factor (TF). The tissue factor is preferably human, and the macromolecular substrate preferably is Factor X (FX) or Factor IX (FIX). In a particularly preferred embodiment, the antibody selected recognizes an epitope which includes a TF region directly interacting with substrate factor FX or FIX, preferably by binding to a site which prevents or blocks association of TF with a Gla domain of the substrate factor. In another preferred embodiment, the antibody selected binds an epitope comprising residues K165, K166 and K201 of hTF. In yet another preferred embodiment, the epitope further comprises residues N199, R200 and I152 of hTF. In a further preferred embodiment, the epitope additionally comprises residue Y156 of hTF. In a particular embodiment, the method is used to identify antibodies that bind essentially to the same hTF epitope as any of antibodies D3, 5G6 and TF8-5G9. In some instances, it might be advantageous to select antibodies that have the binding properties specified above, and do not interfere with the association of hTF and Factor VIIa (FVIIa). All antibodies identified in accordance with the present invention may be poly- or monoclonal antibodies (as hereinafter defined), and may be rodent, (e.g. murine), humanized or human antibodies.
The invention also covers compositions comprising the antibodies identified in accordance with the present invention, and methods of using such antibodies to block a TF-FVIIa mediated or associated process or event, or to prevent or treat a TF-FVIIa related disease or disorder, including but not limited to, thrombotic and coagulopathic disorders.
In another aspect, the invention concerns a method for producing an antibody having enhanced anticoagulant potency, comprising raising antibodies against an antigen comprising at least part of the C-terminal macromolecular substrate binding region of tissue factor (TF). Again, the antibodies may be poly- or monoclonal antibodies (as hereinafter defined), including rodent, e.g. murine, humanized and human antibodies. In a preferred embodiment, the antibodies are raised against an antigen comprising the entire C-terminal macromolecular substrate-binding region of TF, preferably human TF (hTF). Preferably, the antigen used to raise the antibodies comprises residues K165, K166 and K201, and optionally residues N199, R200 and I152 o hTF. The antigen may additionally contain residue Y156 of hTF.
In yet another aspect, the invention concerns an anti-tissue factor (anti-TF) antibody heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1 (VH SEQUENCE OF MURINE D3, FIG. 8) or SEQ ID NO: 2 (VH SEQUENCE OF HUMANIZED D3H44, FIG. 8).
In a further aspect, the invention concerns an anti-tissue factor (anti-TF) light chain variable domain comprising the amino acid sequence of SEQ ID NO: 3 (VL SEQUENCE OF MURINE D3, FIG. 9) or SEQ ID NO: 4 (VL SEQUENCE OF HUMANIZED D3H44, FIG. 9).
In a further aspect, the invention concerns an anti-tissue factor (anti-TF) heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 5 (VH SEQUENCE OF MURINE 5G6xe2x80x94FIG. 15).
In a different aspect, the invention concerns an anti-tissue factor (anti-TF) light chain variable domain comprising the amino acid sequence of SEQ ID NO: 6 (VL SEQUENCE OF MURINE 5G6xe2x80x94FIG. 15).
In another aspect, the invention concerns isolated nucleic acid comprising a sequence encoding an anti-tissue factor (anti-TF) antibody heavy chain variable domain of SEQ ID NO: 1, 2 or 5.
In yet another aspect, the invention concerns isolated nucleic acid comprising a sequence encoding an anti-tissue factor (anti-TF) antibody light chain variable domain of SEQ ID NO: 3, 4 or 6.
In a further aspect, the invention concerns a vector comprising, and capable of expressing, a nucleic acid as hereinabove defined, a recombinant host cell transformed with such vector, a cell culture comprising such recombinant host cell, and a method for expressing said nucleic acid to produce the encoded polypeptide.
The invention also concerns a humanized anti-tissue factor (anti-TF) antibody comprising a heavy and a light chain variable domain, wherein the heavy chain variable domain comprises hypervariable regions CDR-H1 having the sequence of GFNIKEYYMH (SEQ ID NO: 7), CDR-H2 having the sequence of LIDPEQGNTIYDPKFQD (SEQ ID NO: 8) and CDR-H3 having the sequence of DTAAYFDY (SEQ ID NO: 9). In a particular embodiment, the humanized anti-TF antibody of the present invention has a light chain variable domain comprising hypervariable regions CDR-L1 having the sequence of RASRDIKSYLN (SEQ ID NO: 10), CDR-L2 having the sequence of YATSLAE (SEQ ID NO: 11) and CDR-L3 having the sequence of LQHGESPWT (SEQ ID NO: 12). Preferably, both the heavy and light chain hypervariable regions are provided in a human framework region. Particular antibodies that are within the scope of the present invention include, without limitation: (a) murine antibody D3 (D3Mur), (b) humanized antibody D3H44, (c) murine antibody 5G6, and (d) antibodies specifically binding essentially the same epitope as any one of antibodies (a)-(c).
In another aspect, the invention concerns isolated nucleic acid comprising a sequence encoding a humanized anti-TF antibody heavy or light chain variable domain as hereinabove defined, a vector comprising and capable of expressing such nucleic acid, a recombinant host cell transformed with such vector, a cell culture comprising such recombinant host cell, and a method for expressing said nucleic acid to produce the encoded polypeptide.
In another aspect, the invention concerns a composition comprising an anti-tissue factor (anti-TF) antibody identifiable by the method of claim 1, in admixture with a pharmaceutically acceptable carrier. The antibody preferably is an anti-hTF antibody, and is preferably humanized or human. The composition may, for example, comprise an antibody selected from the group consisting of (a) murine antibody D3 (D3Mur), (b) humanized antibody D3H44, (c) murine antibody 5G6, and (d) an antibody specifically binding essentially the same epitope as any one of antibodies (a)-(c), in admixture with a pharmaceutically acceptable carrier.
The invention further concerns a method for the prevention or treatment of a TF-FVIIa related disease or disorder, such as thrombotic or coagulopathic disorder, comprising administering to a subject an effective amount of an anti-tissue factor (anti-TF) antibody of the present invention.
The invention also concerns diagnostic methods, diagnostic kits and articles of manufacture comprising one or more antibodies of the present invention, optionally in combination with one or more further active ingredients useful in the desired diagnostic or therapeutic application.